Prolactin in the brushtail possum (Trichosurus vulpecula): development of homologous radioimmunoassay using recombinant possum prolactin.

We report the production of recombinant possum prolactin (posPrl), and its use in the development and validation of a highly specific homologous radioimmunoassay for the measurement of prolactin (Prl) in brushtail possums. This enabled the subsequent investigation of some basic mechanisms involved in the regulation of Prl secretion in this species. Recombinant posPrl spanning the entire coding region was expressed in Escherichia coli, resulting in a 199 amino acid protein with a molecular weight approximately 23 kDa. The potency of posPrl was 45.3 +/- 4.8% that of ovine Prl in a radioreceptor assay using possum mammary gland receptors and induced a 3.4 +/- 0.8-fold increase in progesterone secretion in primary possum granulosa cells. Antiserum (G27) was raised against recombinant posPrl and was highly specific for possum Prl (approximately 30% binding at 1:60,000 final dilution), and exhibited negligible cross-reactivity (<0.0001%) with possum growth hormone. Serial dilutions of pituitary gland extracts, and plasma samples from male and female possums gave parallel inhibition curves to recombinant posPrl standards in the assay. Biological validation of the RIA included treating possums with drugs known to alter Prl secretion in other mammals. In seasonally anoestrous female possums, administration of 20 microg thyrotropin-releasing hormone (TRH) resulted in a 15-fold increase (P < 0.01) in plasma Prl concentrations. In mid-late lactating female possums, a bolus of cabergoline (dopamine agonist; 75 microg) reduced (P < 0.05) plasma Prl levels to baseline for 24 h, while repeated administration (6 x 75 microg at 12 h intervals) suppressed (P < 0.01) plasma Prl concentrations until 24h after the last injection. Prolonged inhibition of Prl levels subsequently caused marked (P < 0.01) attenuation in rate of bodyweight increase of pouch young. The amplitude of the Prl surge in response to a bolus of TRH (15 microg) was 5-fold lower in cabergoline-treated, compared to control mid-late lactating possums. In conclusion, we report the development and validation of a robust and sensitive RIA for measuring Prl concentrations in the plasma of brushtail possums.

Expression of the FcRn receptor (alpha and beta) gene homologues in the intestine of suckling brushtail possum (Trichosurus vulpecula) pouch young.

The neonatal IgG transporter FcRn consists of two chains, FcRn alpha and beta (also known as beta(2) microglobulin), and is involved in transferring IgG molecules across both mammary and intestinal epithelial cells. Developmental changes in FcRn IgG alpha and beta chain mRNA levels were investigated in the gut of brushtail possum (Trichosurus vulpecula) pouch young (PY) using Northern hybridisation. FcRn alpha transcripts were detected in the PY proximal intestine at all times examined, between days 1 and 195 of post-natal life, with increased levels detected from around day 110. The beta(2) microglobulin transcript levels in the PY proximal intestine were low to undetectable until day 110 of post-natal life and then increased dramatically after day 159. Both the FcRn alpha and beta gene transcripts were detected in a wide range of tissues in the adult possum (>365 days). Genomic sequences located 5' to the start of transcription of the FcRn alpha and beta(2) microglobulin genes were cloned and analysed for predicted cis-acting transcription control elements. Both the FcRn alpha and beta(2) microglobulin genomic sequences contained STAT5 binding motifs consistent with the transcription of both genes being modulated by prolactin. Using in situ hybridisation, the FcRn alpha and beta(2) microglobulin transcripts were localised to the epithelial cells of the PY intestine. However, no prolactin receptor transcripts were detected in the same epithelial cells suggesting that the observed changes in FcRn alpha and beta(2) microglobulin gene expression in the proximal intestine are not modulated directly by prolactin. The results are consistent with the hypothesis that changes in FcRn alpha and beta(2) microglobulin gene expression take place in the possum PY intestine to accommodate changes in maternal milk composition to meet the changing immunological demands of the PY.

Differential expression of the whey acidic protein gene during lactation in the brushtail possum (Trichosurus vulpecula).

The whey acidic protein (WAP) is a whey protein found in the milk of a number of species. We have isolated and characterised a WAP cDNA clone from the brushtail possum (Trichosurus vulpecula) and examined its expression in the mammary gland. The amino acid sequences of WAP from the possum and another marsupial, the tammar wallaby, share 69% identity, however, less sequence identity exists between the marsupial and eutherian WAP sequences (30-37%). The possum and tammar WAP genes consist of three four-disulphide core (4-DSC) domains, with a WAP motif at the beginning of each domain. In contrast, the eutherian WAP sequences consist of two 4-DSC domains with the WAP motif only present in the second domain. This WAP motif is also present in a number of protease inhibitors found in a wide range of species. Phylogenetic analysis of marsupial and eutherian WAP sequences suggests that the ancestral WAP gene has three domains and that one of the domains has been deleted from the eutherian gene. The profile of WAP gene expression in the possum mammary gland changed throughout lactation, with WAP mRNA levels reaching a peak between days 106 and 177 of lactation. The level of WAP mRNA in the mammary gland appeared to be correlated with the level of circulating prolactin in the lactating female and was different to that observed for several other whey protein genes. Overlapping expression of the WAP and early lactation protein genes, both of which are putative protease inhibitors, may provide protection of milk immunoglobulins that are required for the prolonged period of passive immune transfer to the marsupial pouch young.

Expression of the Fc receptor in the mammary gland during lactation in the marsupial Trichosurus vulpecula (brushtail possum).

One of several functions described for the Fc receptor is regulation of IgG isotype transport into milk. The first marsupial homologues of the Fc receptor heavy and light chains, FcRn and beta-2 microglobulin, from the brushtail possum have been cloned and characterised. The level of FcRn mRNA in the possum mammary gland was highest at the start of lactation, and decreased slowly thereafter. Expression of FcRn mRNA did not increase during the switch phase when the concentration of IgG in milk is highest. In contrast, the level of beta-2 microglobulin mRNA in the mammary gland increased during the switch phase when milk IgG concentration also increases. This correlation between beta-2 microglobulin mRNA expression in the mammary gland with the time of active IgG-transfer into milk was also observed in the bovine and murine mammary gland. This suggests that expression of the Fc receptor in the mammary gland is controlled by the expression of beta-2 microglobulin and that its expression is upregulated during the period of highest IgG-transfer into milk.

Valuation of consumption and sale of forest goods from a Central American rain forest

Researchers recognize that society needs accurate and comprehensive estimates of the economic value of rain forests to assess conservation and management options. Valuation of forests can help us to decide whether to implement policies that reconcile the value different groups attach to forests. Here we have measured the value of the rain forest to local populations by monitoring the foods, construction and craft materials, and medicines consumed or sold from the forest by 32 Indian households in two villages in Honduras over 2.5 years. We have directly measured the detailed, comprehensive consumption patterns of rain forest products by an indigenous population and the value of that consumption in local markets. The combined value of consumption and sale of forest goods ranged from US$17.79 to US$23.72 per hectare per year, at the lower end of previous estimates (between US$49 and US$1,089 (mean US$347) per hectare per year). Although outsiders value the rain forest for its high-use and non-use values, local people receive a small share of the total value. Unless rural people are paid for the non-local values of rain forests, they may be easily persuaded to deforest.

Immunological protection of the vulnerable marsupial pouch young: two periods of immune transfer during lactation in Trichosurus vulpecula (brushtail possum).

Marsupial young are born with an underdeveloped immune system and are dependent upon passively acquired immune protection provided by the mother's milk. Colostrum and milk samples were collected from the brushtail possum throughout lactation and the concentration of secretory IgA (sIgA), IgG and transferrin was determined by Western blotting. Two periods of immune transfer were identified. The first, a colostral phase, occurs immediately after birth and involves sIgA, IgG and transferrin. During the early lactation stage, pouch young receive milk of a unique composition as they undergo developmental changes in the pouch that occur in utero for eutherian mammals. At the end of this external gestation, the composition of the milk changes (switch phase) to resemble that of eutherian mammals in the late lactation phase. The second transfer of immunity consists of IgG and transferrin, and occurs during the switch phase prior to maturation of the immune response.

Cloning of a marsupial kappa-casein cDNA from the brushtail possum (Trichosurus vulpecula).

The main role of kappa-casein in milk is to stabilize the formation of casein micelles. Although marsupial milk contains casein micelles, kappa-casein had not been identified in any species. In these experiments, the first marsupial kappa-casein has been prepared as enriched casein fractions and the cDNA cloned from the brushtail possum (Trichosurus vulpecula). Possum kappa-casein is a 158 amino acid peptide that shares low amino acid sequence identity (20-30%) with that of eutherian kappa-caseins. In the gut of suckling young, casein micelles clot when kappa-casein is cleaved by chymosin at a specific site. Eutherian kappa-casein sequences are classified according to the sequence of the chymosin cleavage site: Phe-Met, Phe-Ile or Phe-Leu. Possum kappa-casein appears to form a separate class, with a putative chymosin cleavage site of Phe-Ala, which is different from that found in eutherian mammals. Other features of kappa-caseins, such as the location of the N-terminal cysteine, solubility in the presence of calcium, and the O-glycosylation sites on threonine residues in the C-terminus of the molecule, are conserved in the possum sequence. The kappa-casein gene was expressed throughout lactation in the mammary gland, and although mRNA levels of kappa-, alpha- and beta-casein varied between animals there appeared to be a correlation in the expression of these genes within an individual animal. This suggests that a common transcription regulatory region may be controlling expression of all three genes in the possum.

Prolactin, alone or in combination with glucocorticoids, enhances tight junction formation and expression of the tight junction protein occludin in mammary cells.

Tight junctions (TJ) between adjacent epithelial cells play an important role in maintaining mammary function in the differentiated mammary gland. Mouse mammary cell lines (HC11 and Comma-1D) were used to investigate the effect of the lactogenic hormones prolactin (PRL) and glucocorticoids on the formation of mammary TJ. TJ formation was assessed by an increase in transepithelial electrical resistance and a decrease in paracellular flux of radiolabeled inulin. Both PRL and the synthetic glucocorticoid dexamethasone (DEX) stimulated TJ formation. The biggest effect on TJ formation was observed when both hormones were used in combination, but only when cells were pretreated with DEX. The effects of PRL and DEX are mediated, at least in part, via expression of the transmembrane TJ protein occludin. In summary, these data are the first to show an effect of PRL on mammary TJ formation and the expression of TJ proteins, and confirm the TJ-stimulating effects of glucocorticoids that have been reported previously.

Two stages of increased IgA transfer during lactation in the marsupial, trichosurus vulpecula (Brushtail possum).

The polymeric Ig receptor (pIgR) and J chain molecules are involved in the transfer of IgA across the mammary gland epithelia into milk. The J chain binds two IgA molecules to form dimeric IgA, and the pIgR transports this complex through epithelial cells. We report here the cloning of the first marsupial homologues for the pIgR and J chain from the brushtail possum. Marsupial young are born after a short gestation and are less developed than eutherian newborn. The pouch young is completely dependent on milk as its sole source of nutrition during early lactation and this phase can be considered to be equivalent to an external gestation. Two periods of increased expression of pIgR, J chain, and IgA heavy chain mRNAs were observed in the mammary gland during lactation. The first occurs for a brief period after birth of the pouch young and is likely to reflect IgA transfer via the colostrum. The second period of increased expression, which is unique to marsupials, occurs after the early lactation period and just before young exit the pouch. We propose that this represents a second colostral-like phase at the end of the external gestation.

Cloning and expression of the transferrin and ferritin genes in a marsupial, the brushtail possum (Trichosurus vulpecula).

Transferrin and ferritin cDNAs have been isolated and characterised from the common brushtail possum (Trichosurus vulpecula), the first marsupial examples of these genes. The transferrin cDNA encodes a 711 amino acid pre-protein which shows high levels of amino acid identity with eutherian transferrins (58-60%) and lactoferrins (54-56%). Phylogenetic analysis suggests that the possum transferrin has evolved independently along a pathway distinct from that of the eutherian transferrins and lactoferrins. Possum H-ferritin is a 182 residue protein which shares 86-94% amino acid identity with mammalian, avian and amphibian sequences. Ferritin mRNA was detected in all tissues tested, whereas transferrin was highly expressed in possum liver and mammary gland, and at lower levels in heart, testis and lung. In the possum mammary gland, ferritin mRNA was expressed throughout lactation with higher levels during the first 30 days which coincides with the high iron concentration of milk at this time. The transferrin gene was differentially expressed during lactation with peak mRNA levels detected during the first 6 days of lactation and after day 106 throughout late lactation. The pattern of transferrin mRNA expression in the mammary gland was identical to that of another whey protein, the late lactation protein, suggesting that the transcription of these genes may be regulated by a similar mechanism in this tissue.

The prolactin receptor from the brushtail possum (Trichosurus vulpecula): cDNA cloning, expression and functional analysis.

A full length, prolactin receptor cDNA clone has been isolated from the brushtail possum (Trichosurus vulpecula). This clone encodes a 625 amino acid protein which shares 60-70 and 54% sequence identity with prolactin receptor (long form) sequences from mammalian and avian species, respectively. Sequence similarity was highest in the extra-cellular, hormone-binding domain and in specific regions of the intracellular domain which regulates prolactin receptor signalling in cells. Prolactin receptor mRNA was detected in a wide range of possum tissues and in the mammary gland the PRL-R gene was differentially expressed during lactation with peak mRNA levels being detected during the first 6 days of lactation and after day 115 throughout late lactation. This pattern of PRL-R mRNA expression in the mammary gland is similar to that observed for circulating prolactin in the lactating possum. In CHO cells transiently transfected with the possum prolactin receptor, expression of a beta-lactoglobulin promoter/reporter gene construct was increased 3-fold by adding prolactin. The possum prolactin receptor is therefore capable of binding ovine prolactin and activating the Jak2/Stat5 signalling cascade. This provides evidence for the highly conserved nature of the prolactin signalling pathway in mammalian evolution.

Differential expression of milk protein genes during lactation in the common brushtail possum (Trichosurus vulpecula).

In the common brushtail possum (Trichosurus vulpecula) lactation lasts for 200 days and consists of two distinct phases. Milk composition changes dramatically between phase 2 and 3, which correspond to early and late lactation respectively (phase 1 corresponds to pregnancy). RNA expression patterns have been established for eight major milk protein genes throughout lactation in possum mammary glands. The levels of mRNA expressed from two genes, encoding the early and late lactation proteins, were differentially regulated during lactation, with peak RNA levels occurring in phase 2 and 3 of lactation respectively. Expression of these two RNA transcripts did not overlap, and neither gene was expressed at significant levels between days 116 to 125, suggesting that the transition from phase 2 to phase 3 of lactation occurs at this time. The level of lysozyme, alpha-lactalbumin and trichosurin mRNA increased in phase 3 of lactation, whereas the levels of beta-lactoglobulin, alpha-casein and beta-casein mRNA remained constant throughout lactation. In the non-suckled gland, expression of milk protein genes was greatly reduced by day 6 of lactation. In conclusion, the early and late lactation protein genes are good markers for phase 2 and 3 of lactation, with the transition between these phases occurring around day 120 of lactation in the possum.

Molecular cloning of ERp29, a novel and widely expressed resident of the endoplasmic reticulum.

We have isolated a full-length cDNA clone for a novel 29 kDa protein that is highly expressed in rat enamel cells. The clone encodes a 259-residue protein, here named ERp29, with structural features (signal peptide and a variant endoplasmic reticulum-retention motif, KEEL) that indicate it is a reticuloplasmin. ERp29 has limited homology with protein disulfide isomerase and its cognates, but lacks their characteristic thioredoxin-like catalytic moiety and calcium-binding motifs. ERp29 mRNA was expressed in all rat tissues tested, and a homologous transcript was detected in other animal livers (primate, ruminant, marsupial). In human hepatoma cells, ERp29 mRNA expression was not increased by stresses (tunicamycin, calcium ionophore) that induced other reticuloplasmins. We conclude that ERp29 is a new, highly conserved member of the reticuloplasmin family which is widely expressed. The apparent lack of both calcium binding properties and stress responsiveness distinguish ERp29 from all major reticuloplasmins characterised to dates.

Krox20 may play a key role in the stabilization of long-term potentiation.

Long-term potentiation-inducing stimulation of the perforant path was followed in dentate gyrus granule cells by a dramatic increase of mRNA and protein for Krox20, a zinc-finger-containing transcription factor. Induction of Krox20 required stimulation sufficient to induce LTP and was prevented by NMDA antagonists CPP and MK-801, which block LTP induction. Krox20 protein increased within 20 min of tetanization, was maximal between 1 and 8 h, and was still significantly elevated at 24 h after LTP induction. This prolonged appearance is in striking contrast with the more transient induction of the related molecule, Krox24. The elevation in the mRNA for Krox20 and Krox24 was of similar duration, suggesting that the Krox20 protein has a greater stability and may play a key role in the stabilization of long-term potentiation.

The proximal milk protein binding factor binding site is required for the prolactin responsiveness of the sheep beta-lactoglobulin promoter in Chinese hamster ovary cells.

To identify cis-acting prolactin (PRL) response elements within the sheep beta-lactoglobulin (BLG) promoter, CHO cells were co-transfected with a rabbit PRL-receptor (PRL-R) expression plasmid and a number of BLG-CAT constructs. Resection through the 4200 bp BLG promoter diminished the PRL response. Mutation of the proximal binding site for milk protein binding factor (MPBF), a previously described mammary gland transcription factor, abolished the PRL inducibility of full length and shorter forms of the promoter. MPBF was shown to be similar to the Stat protein mammary gland factor (MGF) which has been shown to mediate PRL responsiveness of the rat beta-casein gene in mammary cells. MPBF binding activity was detected in the nucleus of CHO cells and was increased 2-6-fold in cells stably transfected with the PRL-R. The lactating mammary gland has high levels of MPBF binding activity and it is likely that this has an important role in the PRL induction of a variety of milk protein genes.

The mammary factor MPBF is a prolactin-induced transcriptional regulator which binds to STAT factor recognition sites.

Site-directed mutagenesis of the three binding sites for the mammary factor MPBF in the beta-lactoglobulin (BLG) promoter demonstrates that MPBF is a transcriptional activator of the BLG gene in mammary cells. MPBF requires phosphorylation on tyrosine for maximum binding activity and binds to GAS (interferon gamma-activation site) elements which are similar to the MPBF binding sites. Prolactin induces MPBF binding activity in CHO cells and is not antigenically related to Stat1 (p91) and Stat2 (p113), suggesting that this transcription factor is likely to be another member of the STAT family of cytokine/growth factor-induced transcription factors.

Correlations between immediate early gene induction and the persistence of long-term potentiation.

The duration of long-term potentiation in the dentate gyrus of awake rats was examined following systematic manipulation of the number of stimulus trains delivered. This was correlated with the induction of immediate early genes in separate groups of animals given identical stimulus regimes. Following 10 trains of stimulation, long-term potentiation decayed with a time constant of up to several days (long-term potentiation 2), and this correlated with the appearance of an increase in the messenger RNA and protein levels of zif/268. Increasing the number of stimulus trains resulted in a greater probability of eliciting long-term potentiation with a time constant of several weeks (long-term potentiation 3), as well as increasing the induction of zif/268, c-Jun, Jun-B, Jun-D and Fos-related proteins. When 10 trains were delivered repeatedly on up to five consecutive days, only the zif/268 protein levels showed associated changes. These data provide support for the hypothesis that long-term potentiation 3 involves mechanisms additional to those for long-term potentiation 2. One possible mechanism is altered gene expression, initiated by immediate early gene transcription factors such as zif/268 and possibly homo- or heterodimers of Fos and Jun family members, that then contributes to the stabilization or maintenance of long-term potentiation 3.

Characterization of two sheep insulin-like growth factor II cDNAs with different 5'-untranslated regions.

We have isolated two sheep IGF-II clones which have the same coding regions but have different 5'-untranslated regions. There is no homology between these 5'-UTR sequences which are homologous to exons 5 and 6 of the human IGFII gene. In humans these exons are transcribed only during fetal development, but in the sheep these transcripts were detected up to 28 weeks after parturition. The sizes of the sheep IGF-II mRNA transcripts are the same as those observed for the human gene, suggesting structural and transcriptional similarity between the human and sheep IGF-II genes. The sheep could therefore be a good model system in which to study IGF-II expression.

Differential expression of immediate early genes after hippocampal long-term potentiation in awake rats.

The pattern of expression of fos and jun family immediate early genes following the induction of long-term potentiation (LTP) was investigated in the dentate gyrus of awake rats. Rapid, transient increases in the levels of c-jun and jun-B mRNA and protein, and in the levels of Fos-related proteins (FRAs), occurred in the dentate gyrus after LTP-inducing tetanization of the perforant path. A delayed, and more prolonged induction occurred for jun-D mRNA and protein. The induction of c-Jun, Jun-B, Jun-D and Fos-related proteins was prevented by administration of an N-methyl-D-aspartate receptor antagonist, which also blocked LTP induction, and by pentobarbital, which reduced but did not block LTP. These findings show that differential expression of fos and jun gene family members occurs in a distinct pattern following LTP in awake rats. The responsive genes may participate in the biochemical cascade leading to the long-term stabilization of synaptic modifications.